Recent advances in cancer proteomics have allowed for systemic identification of the phosphorylation events associated with cancer progression. However, validating and translating these large datasets from in vitro to in vivo systems remains a major challenge. The long-term goal of our research is to understand how the protein phosphorylation events induced by Abelson (ABL) tyrosine kinases contribute to breast cancer development. The objective of this exploratory study is to develop a streamlined model system for rapid in vivo analysis of the function of ABL-mediated protein phosphorylation in breast cancer growth and metastasis. We have recently developed a strategy to conditionally replace an endogenous gene expression with the expression of its mutant counterpart by using short hairpin RNA-based technology. We hypothesize that, by using this strategy combined with the recombination-mediated cassette exchange (RMCE) technology, a streamlined system can be developed to provide a fast, reversible, and cost-effective way to analyze the function of a given phosphorylation-deficient or -mimetic mutant in breast cancer development. We will test this hypothesis by defining the role of tyrosine phosphorylation of ABL interactor 1 (Abi1), an ABL substrate and key regulator of actin cytoskeleton remodeling, in breast cancer development. The rationale behind this research is that once this system is developed, we will be able to quickly analyze the function of a large set of ABL-mediated phosphorylation events obtained from cancer proteomic studies and identify those that are essential for breast cancer development for prognostic and therapeutic purposes. In specific aim 1, a set of conditional Abi1 gene silencing/re-expressing cassettes will be developed to analyze how ABL-mediated Abi1 tyrosine phosphorylation affects breast cancer cell function and signaling. In aim 2, how the ABL-mediated Abi1 phosphorylation contributes to breast cancer progression in vivo will be determined by using conditional gene replacement strategy combined with a brain metastasis mouse model recently developed in our lab. This project is innovative because it proposes a new strategy different from conventional gene knockin for in vivo protein mutation analysis. If successful, this line of research will enable a high-throughput function analysis of protein mutation in breast cancer development. Identifying ABL-mediated phosphorylation events essential for breast cancer development will not only help us to understand how ABL kinases contribute to breast cancer development, but also provide prognostic markers for selection of breast cancer patients who may benefit from targeted molecular therapy.